1. Field of the Invention
This invention relates generally to methods and compositions for inhibiting .beta.-amyloid peptide (.beta.AP) production in cells. In particular, this invention relates to compounds which are capable of inhibiting the intracellular production of .beta.AP, and the use of such compounds in methods for inhibiting .beta.AP production.
This invention also relates to an isolated novel protein, Cathepsin Y, which is a novel carboxypeptidase involved in the generation of .beta.AP. Methods for isolation of this protein are provided. DNA isolates coding for Cathepsin Y and methods of obtaining such DNA are provided, together with expression systems for recombinant production of Cathepsin Y useful in therapeutic or diagnostic compositions.
2. State of the Art
Alzheimer's Disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and, ultimately, death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms and course is currently known.
The brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restricted anatomical distribution are also found in the brains of most aged humans who do not have clinical AD. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D). At present, a definitive diagnosis of AD usually requires observing the aforementioned lesions in the brain tissue of patients who have died with the disease or, rarely, in small biopsied samples of brain tissue taken during an invasive neuro-surgical procedure.
The principal chemical constituent of the amyloid plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of AD and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the .beta.-amyloid peptide (.beta.AP) or sometimes A.beta., A.beta.P or ,.beta./A4. .beta.AP is a fragment of a large membrane-spanning glycoprotein, referred to herein as the .beta.-amyloid precursor protein (APP), comprising approximately 39-43 amino acid residues. This protein fragment was first purified, and a partial amino acid sequence was reported in, Glenner and Wong, Biochem. Biophys. Res. Commun. 120:885-890 (1984). The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829.
.beta.AP is further characterized by its relative mobility in SDS-polyacrylamide gel electrophoresis or high performance liquid chromatography (HPLC). .beta.AP can occur in a filamentous polymeric form which exhibits the Congo-red and thioflavin-S dye-binding characteristics of amyloid. .beta.AP can also occur in a non-filamentous form ("preamyloid" or "amorphous" or "diffuse" deposits) in tissue, in which form no detectable birefringent staining by Congo red occurs. A portion of this protein in the insoluble form obtained from meningeal blood vessels is described in U.S. Pat. No. 4,666,829.
APP is normally produced by cells in many tissues of various animals, including humans. APP is encoded by a gene on the long arm of human chromosome 21. Knowledge of the structure of the gene encoding APP has indicated that .beta.AP arises as a peptide fragment from cleavage of APP by at least one heretofore unidentified protease. This cleavage appears to occur in the lysosomes. The precise biochemical pathway by which the .beta.AP fragment is cleaved from the APP and subsequently deposited as amyloid plaques is still under investigation.
Several lines of evidence indicate that progressive cerebral deposition of .beta.AP plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades (for a review, see Selkoe, (1991) Neuron 6:487). Recently, it has been shown that .beta.AP is released from neural cells grown in culture as well as into cerebral spinal fluid of both normal individuals and AD patients.
Certain inherited mutations which occur in the APP gene are also known to cause AD and AD-related conditions. For example, mis-sense DNA mutations at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not in unaffected members of several families with a genetically determined (familial) form of AD (Goate et al., Nature 349:704-706 (1991); Chartier Harlan et al., Nature 353:844-846 (1991); and Murrell et al., (1991) Science 254:97-99). A double mutation changing lysine.sup.595 -methionine.sup.596 to asparagine.sup.595 -leucinell.sup.596 (with reference to the 695-amino acid isoform of APP) found in a Swedish family was reported in 1992 (Mullan et al., (1992) Nature Genet 1:345-347) and is referred to as the Swedish variant or mutation.
Genetic linkage analyses have demonstrated that these mutations, as well as certain other mutations in the APP gene, are the specific molecular cause of AD in the affected members of such families. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of APP has been identified as the cause of the .beta.AP deposition disease, HCHWA-D, and a change from alanine to glycine at amino acid 692 appears to cause a phenotype that resembles AD in some patients but HCHWA-D in others. See, Younkin, et al., Science 259:514-516 (1993).
Despite the progress which has been made in understanding underlying mechanisms of AD and other .beta.AP-related diseases, there remains a need to develop compositions and methods for treatment of the disease(s).